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Different Oncogenic KRAS Mutations Produce Distinct Heterogeneous Outcomes In Signaling Pathways Of Isogenic Mouse Embryonic Fibroblasts

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dc.contributor.advisor Pierobon, Mariaelena El Gazzah, Emna
dc.creator El Gazzah, Emna 2021-04-29 2021-10-14T13:23:37Z 2021-10-14T13:23:37Z
dc.description.abstract RAS proteins are a family of small GTPases, they are central to transduction of mitogenic signals that control cell growth, proliferation, survival and metabolism. A gain of function mutation in this family of proteins, either by increased expression or activation state, provides the cell with sustained proliferative signaling, one of cancers basic eight hallmarks. For that reason, RAS proteins are one of the most frequently mutated oncogenes, mutated in up to 85% of all human cancers. of the three RAS isoforms: HRAS, NRAS and KRAS, KRAS astoundingly makes up to 85% of all RAS mutations. 99.2% of KRAS mutations occur at three distinct codon hotspots: G12, G13 and Q61, with mutations at codon G12 making up 90% of these. Moreover, compiling evidence points towards the notion that not all KRAS mutations act equally. There is a clear imbalance in the frequency of KRAS mutations that appear not only across different cancer types but among the same type too. There is a strong correlation between the appearance of a distinct mutation and cancer type, histology and carcinogen. These context specific trends really reflect the underlying complexity behind the different mutations. An important set of players that regulate KRAS signal transduction pathways and define their specificity are the scaffold proteins. They regulate signal duration, strength and insulation through dynamic scaffold-kinase interactions (SKIs). SKIs have a fundamental influence on signal transduction and so for the different KRAS mutants to produce distinct effects, they must be able to manipulate this. In this study, we aim to explore broad changes in signal transduction across different KRAS mutations and to evaluate how different mutations can affect SKIs. To study KRAS mutant specific effect on cell signaling networks, we used isogenic Mouse Embryonic Fibroblasts (MEFs) engineered to selectively harbor and express 6 KRAS oncogenic variants. We first used a multiplex, high throughput immunoassay, Reverse Phase Protein Microarray (RPPA), to capture broad signaling changes across our cell lines. To further explore the effect of KRAS mutations on downstream signaling events, we characterized and compared functional scaffold-kinase interactions across models harboring mutations affecting codon 12. Scaffold-kinase interactions were analyzed using the newly developed Multi-nodal Protein Interactome Network Array (MPINA) which combines serial Co-immunoprecipitation (Co-IP) with RPPA to selectively isolate protein complexes and identify the activation status of their constituents. Broad signaling profile of the 6 models has shown heterogeneous and mutationspecific signaling activity. Overall, mutations at codon 12 cluster together exhibiting higher signaling activity compared to all models and presenting with the least intricate interconnection network. On the contrary, mutations of codon 61 presented with the least active signaling dynamics and interestingly interconnection network with the highest complexity. Furthermore, comparing mutations at codon 12 displayed a clear division into cells with the highest active signaling; G12D and G12C, and cell lines with the lowest active signaling; G12V and G12R. This trend of mutation-specific signaling activity follows through and can be seen across downstream SKI dynamics of mutations at codon 12. Remarkably, we observe the highest difference in SKI dynamics among the two cell lines with highest signaling activities. G12C mutations presented with the most active SKI events while the G12D presented with significantly reduced SKI action. Taken together our data indicate that not all KRAS mutations are equal as downstream signaling nodes are differentially activated based on the type of mutation. Exploring SKI of KRAS downstream signaling molecules may provide additional information of how signaling networks are rearranged in the presence of a KRAS mutation. en_US
dc.language.iso en en_US
dc.title Different Oncogenic KRAS Mutations Produce Distinct Heterogeneous Outcomes In Signaling Pathways Of Isogenic Mouse Embryonic Fibroblasts en_US
dc.type Thesis en_US Master of Science in Biology en_US Master's en_US Biology en_US George Mason University en_US

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